Sensitive quantification of dipicolinic acid from bacterial endospores in soils and sediments

Endospore-forming bacteria make up an important and numerically significant component of microbial communities in a range of settings including soils, industry, hospitals and marine sediments extending into the deep subsurface. Bacterial endospores are non-reproductive structures that protect DNA and improve cell survival during periods unfavourable for bacterial growth. An important determinant of endospores withstanding extreme environmental conditions is 2,6-pyridine dicarboxylic acid (i.e. dipicolinic acid, or DPA), which contributes heat resistance. This study presents an improved HPLC-fluorescence method for DPA quantification using a single 10-min run with pre-column Tb³⁺ chelation. Relative to existing DPA quantification methods, specific improvements pertain to sensitivity, detection limit and range, as well as the development of new free DPA and spore-specific DPA proxies. The method distinguishes DPA from intact and recently germinated spores, enabling responses to germinants in natural samples or experiments to be assessed in a new way. DPA-based endospore quantification depends on accurate spore-specific DPA contents, in particular, thermophilic spores are shown to have a higher DPA content, meaning that marine sediments with plentiful thermophilic spores may require spore number estimates to be revisited. This method has a wide range of potential applications for more accurately quantifying bacterial endospores in diverse environmental samples.     

Spore dipicolinic acid contents used for estimating the number of endospores in sediments

Endospores are heat-resistant bacterial resting stages that can remain viable for long periods of time and may thus accumulate in sediments as a function of sediment age. The number of spores in sediments has only rarely been quantified, because of methodological problems, and consequently little is known about the quantitative contribution of endospores to the total number of prokaryotic cells. We here report on a protocol to determine the number of endospores in sediments and cultures. The method is based on the fluorimetric determination of dipicolinic acid (DPA), a spore core-specific compound, after reaction with terbium chloride. The concentration of DPA in natural samples is converted into endospore numbers using endospore-forming pure cultures as standards. Quenching of the fluorescence by sediment constituents and background fluorescence due to humic substances hampered direct determination of DPA in sediments. To overcome those interferences, DPA was extracted using ethyl acetate prior to fluorimetric measurements of DPA concentrations. The first results indicated that endospore numbers obtained with this method are orders of magnitude higher than numbers obtained by cultivation after pasteurization. In one of the explored sediment cores, endospores accounted for 3% of all stainable prokaryotic cells.     

Genetic relationship between Sarcina ureae and members of the genus Bacillus

Deoxyribonucleic acid-ribonucleic acid (DNA-RNA) and DNA-DNA hybridization studies were performed to determine the degree of genetic relatedness between Sarcina ureae and several members of the genus Bacillus. DNA-RNA hybridization showed a high degree of homology between S. ureae RNA and DNA from Bacillus species having a similar guanine plus cytosine content. The DNA from other genera of the family Micrococcaceae showed less homology with S. ureae RNA than did that of the Bacillus species tested; however, this homology was not found between the DNA of S. ureae and DNA from these Bacillus species or DNA from the other Micrococcaceae tested. Transformation with Bacillus DNA, infection with representatives from several major classes of Bacillus phages, and electrophoretic analysis of proteins in crude extracts of these strains were also attempted as a further test of the genetic relationship between the genera. These experiments did not support the belief that the two groups are closely related genetically.     

On the chromatographic behavior of dipicolinic acid

Summary Paper chromatography of 100g or more of dipicolinic acid (DPA) in acetic acid-containing solvents resulted in 3 distinct ultraviolet-absorbing sports at different R_f values. The ultraviolet absorption spectra of eluates of the respective spots were different; this is ascribed to metal chelates of DPA. As DPA moves up the paper, it chelates with metal impurities in the chromatographic paper. The chelates have lower R_f values than free DPA which is the fastest moving spot. This condition arises only when the amount of DPA exceeds the chelation capacity of metal impurities in the system. Chromatographic paper pre-washed with Na ethylenediamine tetraacetate to remove the metal impurities gave only a single spot. Chromatography in a formic acid-containing solvent did not produce the chelate spots. A possible explanation is advanced for this behavior. A sensitive, simple spectrophotometric assay of DPA is described based on optical density at the wave length of maximal absorption of DPA, 273 m.Professor Dr. A. Rippel zum 70. Geburtstag.International Cooperation Administration Fellow, 1955–57.     

A highly sensitive HPLC method for determination of nanomolar concentrations of dipicolinic acid, a characteristic constituent of bacterial endospores

A high-performance liquid chromatographic method with indirect fluorescence detection has been developed for quantification of dipicolinic acid, a major constituent of bacterial endospores. After separation on a reversed-phase column, a post-column reagent of sodium acetate at 1 mol l(-1) with 50 micromol l(-1) terbium chloride was added for complexation of dipicolinic acid. Terbium monodipicolinate complexes formed were quantified by measuring the fluorescence emission maximum at 548 nm after excitation with UV light at 270 nm wavelength. Parameters of post-column complexation were optimized to achieve a detection limit of 0.5 nmol DPA l(-1), corresponding to about 10(3) Desulfosporosinus orientis endospores per ml. The method was applied to the analysis of spore contamination in tuna and for estimating the endospore numbers in marine sediments.     

An efficient procedure for the isolation of PCR-competent DNA from Bacillus endospores germinated in soil

DNA extraction techniques for endospore-forming bacteria in soil are often labour-intensive and unreliable. Our objective in this work was to investigate whether good quality DNA could be obtained from spores germinated in soil. To this end, endospores from Bacillus subtilis, B. megaterium and B. thuringiensis were inoculated into soil microcosms and germination was induced by addition of LB medium supplemented with l-alanine, glucose, fructose and KCl. Heat resistance count was reduced to 80% for B. subtilis and more than 90% for B. thuringiensis and B. megaterium after a few minutes. Isolation of DNA from soil with a procedure which did not work on spores was shown to be as efficient for in situ-germinated spores as for inoculated vegetative cells. Furthermore, we developed a simple procedure that allowed us to use the recovered DNA in PCR amplifications. The present methodology is simple and efficient; it avoids the use of special equipment and harsh spore rupturing methods and can be carried out with multiple samples.     

Deoxyribonucleic acid base composition of Sporosarcina ureae

Summary The guanine + cytosine (GC) content of the DNAs of 11 cultures of Sporosarcina ureae and one culture of Bacillus pasteurii was determined using the methods of Marmur and Doty (1962), Frédéricq et al. (1961), and paper chromatography. The GC content in DNA of Sporosarcina ureae ranges from 39.3 to 44%. Bacillus pasteurii contained 40.4% GC in DNA. Our results support the opinion that Sporosarcina ureae is related both to non-sporeforming flagellated cocci of the genus Planococcus and to bacilli, particularly to Bacillus pasteurii.     

Activation of an nadph oxidase from maize by dipicolinic acid

An NADPH oxidase has been partially purified from maize kernels. The activity of this enzyme, as measured by NADPH disappearance or oxygen consumption, was enhanced by dipicolinic acid (pyridine 2,6-dicarboxylic acid). Dipicolinic acid was not consumed in this reaction indicating that it was an activator rather than a substrate of this enzyme.     

Fermentative production of dipicolinic acid by Penicilium citreoviride from sugarcane molasses

In Penicillium citreoviride strain 3114, dipicolinic acid (DPA) synthesis is inhibited by Ca⁺⁺ ions and susceptible to catabolite repression, making it unsuitable for fermentation in sugarcane molasses. A mutant, 27133-dpa-Ca-14, was derived through stepwise mutation and selection to produce DPA in the presence of 1000 ppm Ca⁺⁺ and also to be resistant to catabolite repression. With this mutant, higher product concentrations (36 g DPA/l) could be reached without prior removal of Ca⁺⁺ from the molasses. The DPA yields increased by about four times (0.4 g DPA/g glucose consumed) and productivity by two and a half-times (3.0 g DPA/l.d) compared with that of the parent strain 3114. Higher product yields (0.58–0.59 g DPA/g glucose consumed) were obtained in a multiple stage fermentation system. DPA was recovered through sepration by ion exchange chromatography followed by concentration and crystallization.     

Isolation of dipicolinic acid as an insecticidal toxin from Paecilomyces fumosoroseus

Several entomopathogenic fungi produce toxins that could be used as bioinsecticides in integrated pest management programs. Paecilomyces fumosoroseus is currently used for the biological control of the whiteflies Bemisia tabaci and B. argentifolii. Supernatants from submerged batch culture, where the fungus produced abundant dispersed mycelium, conidia and blastospores, were toxic to the whitefly nymphs. The most abundant metabolite was purified by HPLC and identified by mass spectrometry and NMR as dipicolinic acid. Both the dipicolinic acid produced by the fungus and the chemically synthesized compound had insecticidal activity against third-instar nymphs of the insect. Dipicolinic acid was toxic to the whitefly nymphs in bioassays involving topical applications. In submerged culture, the specific growth rate of P. fumosoroseus was 0.054 h⁻¹, the specific glucose consumption rate was 0.1195 g g⁻¹ h⁻¹ and the specific dipicolinic acid production rate was 0.00012 g g⁻¹ h⁻¹. Dipicolinic acid was detected after 24 h when the fungus started growing; and dipicolinic acid production was directly correlated with fungal growth. Nevertheless, the yield was low and the maximal concentration was only 0.041 g l⁻¹. The maximal concentrations of conidia and blastospores (per milliliter) were 1.4×10⁸ and 7×10⁷, respectively.     

Characterization of urease from Sporosarcina ureae

Alkaline stable (pH 7.75–12.5) urease from Sporosarcina ureae was purified over 400-fold by ion exchange and hydrophobic interaction chromatography. The cytoplasmic enzyme was remarkably active with a specific activity of greater than 9300 μmol urea degraded min^-1 mg protein^-1 at pH 7.5, where it has optimal activity. Although S. ureae is closely related to Bacillus pasteurii, known to posses a homopolymeric urease containing 1 nickel per subunit [M _r=65000], the S. ureae enzyme is comprised of three subunits [apparent M _r=63100 (α), 14500 (β), and 8500 (γ)] in an estimated ∝βγ stoichiometry and contains 2.1±0.6 nickel ions per ∝βγ unit as measured by atomic absorption spectrometry. Stationary phase cultures sometimes possessed low levels of urease activity, but the specific activity of cell extracts of partially purified urease preparations from such cultures could be elevated by heat treatment, dilution, or dialysis to values comparable to those observed in samples from exponentially grown cells.